Methylation and Demethylation of DNA in the Higher Animal

在这个项目中，我的目标是阐明脊椎动物染色体DNA的DNA甲基化的调控。为此，我进行了以下实验，并得到了如下结果。我从奇肯(鸟类)和非洲爪哇(两栖动物)中提取了DNA甲基转移酶(MTase)基因，并测定了其核苷酸序列。在培养细胞中表达了这两种基因，并对其性质进行了分析。过表达的小鼠MTase显示出比鸡MTase更高的从头甲基化活性。我已经鉴定出一种新的DNA结合蛋白MMBP-3，它能特异性识别c-Myc结合基序的甲基化形式。MMBP-3的DNA结合活性在增殖细胞中较高，当细胞增殖受阻时，其DNA结合活性降低。在成肌细胞中过表达小鼠MTase可加速肌管的形成。这是由于在增殖条件下直接诱导MyoD基因转录所致。MyoD基因的表达与转录起始点附近特定CpG序列的甲基化呈正相关。我已经分离到一个新的基因，命名为AZ1，它的表达是用去甲基化试剂诱导的。AZ1蛋白定位于精母细胞顶体前。

In the project, I aimed to shed light on the regulation of the DNA methylation of chromosomal DNA in vertebrate. For this purpose, I engaged in the following experiments and got the results as follows.1. I have isolated DNA methyltransferase (MTase) cDNAs from chiken (avian) and Xenopus laevis (amphibian) and determined the nucleotide sequences. Both cDNAs were expressed in cultivated cells and their properties were analyzes. Overexpressed mouse MTase showed higher de novo methylation activity than chicken MTase.2. I have identified a novel DNA binding protein, MMBP-3, that specifically recognizes the methylated form of c-Myc binding motif. The DNA binding activity of the MMBP-3 is high in the proliferating cells and decreases when the cell proliferation is arrested.3. Overexpression of mouse MTase in myoblasts accelerates the myotube formation. This is caused by direct induction of MyoD gene transcription under proliferating conditions. The expression of MyoD gene positively correlates with the methylation of specific CpG sequences near transcriptional start site.4. I have isolated a novel cDNA named AZ1, of which expression is induced with demethylating reagent. The AZ1 protein is localized to the pre-acrosome of spermatocytes.